Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disease characterized by progressive muscle weakness over a patient's lifetime [1]. It is the most common childhood form of muscular dystrophy affecting about 1 out of 3500 live male births worldwide [2]. DMD is primarily caused by out of frame multi-exon deletions in the DMD gene that ablate dystrophin protein production [3]. Dystrophin is an essential component of the dystrophin glycoprotein complex (DGC), which functions in linking the actin cytoskeleton to extracellular matrix to provide sarcolemmal stability in the context of muscle contraction. The DGC also plays a role recruiting and organizing signal transducers at the sarcolemmal membrane. Both of these activities are required for muscle cell health, and thus the absence of dystrophin leads to progressive loss of muscle function. Dystrophin binds to actin via N-terminal sequences and to b dystroglycan within the DGC via carboxyl terminal domains, whereas the central portion of the protein consists of a rod domain containing multiple spectrin repeats. Deletions within the central rod domain that preserve the reading frame can produce an internally deleted dystrophin protein that retains some functionality and localizes to the membrane within the DGC [4]. Typically, the more mild allelic disorder, Becker muscular dystrophy, results from DMD mutations in the rod domain which remain in—frame 3′ of the deletion and produce a functional dystrophin protein [5]. There are no curative therapies for DMD, and the only demonstrated pharmacological treatment is corticosteroids, which may prolong ambulation for up to 3 years, but with substantial side effects [6]. An emerging therapy, exon skipping, targets individual exons with antisense oligos (AOs) for exclusion from mRNA based on an individual's known genomic DNA mutation with the goal to change out-of-frame mutations into in-frame DMD deletions that restore the reading frame in dystrophin mRNA and allow translation of dystrophin protein. FIG. 14 is a schematic illustration of antisense-mediated therapeutic exon skipping. AOs have been successfully demonstrated to promote DMD exon skipping and restore dystrophin protein expression in mice, dogs and humans in recent clinical trials [7-12]. High dose, chronic administration of an exon 23 directed AO in the mdx mouse demonstrated substantial disease reduction highlighting the tremendous promise of this therapy for DMD in humans [13]. A series of AOs are under development for human use and about half of all DMD patients could be treated with the targeting of 6 different exons (51, 45, 53, 44, 52, 50) in the most frequently deleted portion of the gene between exons 45-53 [14]. For instance, DMD exon 51 skipping will be appropriate for about 13% of all DMD patients, and is the first in clinical trials with two different backbone chemistries, 2′-O-methyl phosphorothioate and morpholino phosphorodiamidate (PMO), both of which have shown promising results [8-10]. These studies are paving the way in personalized genetic medicine.
Recent phase 1-2a clinical trial results utilizing systemic 2′-O-methyl modified AO directed against DMD exon 51 (Pro051) rescued dystrophin protein at levels ranging from 1.8-15.6% of normal [8]. A modest improvement in the 6 minute walk test at 48 weeks was observed with weekly subcutaneous dosing of 6 mg/kg in a non-placebo controlled extension trail, but it remains to be determined if the levels of dystophin produced are sufficient to impart substantial functional utility or longterm protection of muscle [15]. Morpholino AO directed against exon 51 (AVI-4658) resulted in dystrophin rescue with up to 55% of myofibers induced to be dystrophin positive after 12 weeks of therapy in humans. However, the total amount of dystrophin induced was generally low, at 0-27% of normal [16]. Further, DMD exon skipping efficacy and dystrophin expression varies across patients, and muscle types.
There is a need for an improvement in exon skipping therapy that would result in more total dystrophin expression and broader effect in multiple muscle groups. For example, synergistic treatments that would permit equal efficacy with reduced AO dose, accompanied by lower toxicity, could substantially impact the practicality of the chronic administration of expensive to produce oligonucleotides [17].